Immobilized enzyme products, especially immobilized enzyme products intended for use in a column has been a rapidly growing field as of the date hereof. Research efforts have been directed towards producing immobilized enzyme products of ever lower price, better physical strength, higher unit activity and of particle size and shapes giving rise to a minimum pressure drop during column operation as well as a high particle strength against abrasion. As of the date hereof, workers in the art have made available a substantial number of reasonably satisfactory methods to immobilize enzymes.
This invention is directed, in a preferred mode thereof, to the conversion of cell bound microbial enzymes into particle form immobilized enzymes made from the cell mass of the microorganism. The discussion of enzyme immobilization hereinafter provided is largely within a context of this type of immobilized enzyme product.
On the whole, as the art has advanced, product and method deficiencies, such as non-optimum particle size distribution, lack of control over particle shape and the cost factor of relatively low product yield, long considered to be unimportant defects in the immobilization process become major defects, which must be obviated. For example, glutaraldehyde has been employed in commercial practice for cross-linking cell bound enzymes according to the teachings of Amotz U.S. Pat. No. 3,980,521. Yet, glutaraldehyde is not an ideal cross-linking agent, for cell bound enzymes at least, reacting only with --NH.sub.2 and --SH groups. The cells of many microorganism species react poorly with glutaraldehyde.
A polyazetidine prepolymer may be employed advantageously for cross-linking purposes, such being suggested by Wood et al. U.S. Pat. No. 4,436,813 and by "A Novel Method of Immobilization and Its Use in Aspartic Acid Production," Wood et al., Bio/Technology, December 1984, pp. 1081-1084. This Patent and Paper ar incorporated by reference herein. The polyazetidine prepolymer cross-linking system is more widely applicable to immobilization of cell bound enzymes than is glutaraldehyde because cross-linking reactions take place between the polyazetidine prepolymer and --COOH and --OH groups as well as --NH.sub.2 and --SH groups.
The instances to which practice of this invention is directed in particular are those when the desired enzyme form constitutes particles made from the microorganism cells, and cellular substances, and cross-linking reagent(s), and optionally, auxiliary cross-linking agents, e.g., proteins and/or agglomerating agents, e.g., polyelectrolytes, and/or finely-divided filler materials. The particles are essentially homogeneous. Such enzyme product form are variously termed herein as cell mass particles and/or cell mass particulate form. It is noted parenthetically, that the process of above-referenced Wood et al. Patent and Paper is directed principally to immobilizing the enzymatically active microorganism cells and cellular substances on carrier particles, and that the inventors hereof strongly prefer the cell mass particle form over a carrier base particle form of immobilized enzyme product the latter not being essentially homogeneous particles.
Efforts by the inventors hereof to employ polyazetidine prepolymer cross-linking to generate cell mass particulate form enzyme products evidenced existence of material deficiencies to the process taught by the prior art. The reaction mixture constitutes an aqueous dispersion of the polyazetidine prepolymer in solution and individual microorganism cells along with any cellular substances present, or other biological material necessitating conduct of the curing reaction en mass. By crushing the reaction product and sieving, a desired particle size fraction may be recovered, but overall the yield of the wanted particle size fraction is usually low, and also, the shape of the individual particles is not controlled. Thus, cross-linking a non-particulate cell mass composition gives rise to immobilized enzyme product wherein particle shape and size is not controlled.
Although the foregoing discussion of the background of the invention and the description of the invention which now follows is couched in terms of cell bound enzymes and a cell mass particulate product, such is done to facilitate understanding of the invention and to describe preferred practice of the invention in fulsome fashion. It is emphasized that practice of the invention is applicable to biological materials more generally, including notably, homogenized cell sludge, enzymes in solution (e.g., extra cellular enzymes), co-enzymes and anti-bodies.